Spider Egg Case Core Fibers: Trimeric Complexes Assembled from TuSp1, ECP-1, and ECP-2

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• 作者：Xiaoyi Hu ; Kristin Kohler ; Arnold M. Falick ; Anne M. F. Moore ; Patrick R. Jones ; and Craig Vierra
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：March 21, 2006
• 年：2006
• 卷：45
• 期：11
• 页码：3506 - 3516
• 全文大小：479K
• 年卷期：v.45,no.11(March 21, 2006)
• ISSN：1520-4995

文摘

Spider silk proteins are well-known for their extraordinary mechanical properties, displayingremarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI)tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate anew cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider,Latrodectus hesperus. Analysis of the primary sequence of this protein reveals ~52% identity to the eggcase protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequenceand expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 andECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 aminoacids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primarysequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GArepeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressedin the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher.MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymaticdigestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containingtubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence forTuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. Incombination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localizationwith TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural rolesin the egg case silk fiber.